Umbilical cord blood-derived stem cells (NiCord) are expanded by Nicotinamide (NAM) technology. NAM is a form of vitamin B3 and a potent inhibitor of enzymes that use nicotinamide adenine dinucleotide (NAD) as a co-factor for their activity, and is linked to the redox state and metabolism of cells. NAM delays differentiation and increases engraftment efficacy of cord blood-derived human CD34(+) cells cultured with cytokines. NAM enables expansion of functional cells in culture, while attenuating the continuous morphological and functional alterations cells typically undergo during ex vivo cultures.
Cells were obtained from UCB samples harvested from consenting mothers after normal full-term deliveries. Samples were collected and frozen. Before use, cells were thawed, the mononuclear cells purified on a Ficoll-Hypaque gradient, and CD34+ cells were isolated using a MidiMACS CD34 Progenitor Cell Isolation Kit.
Purified CD34+ cells were cultured in culture bags at 1 ×104 cells/mL (at least 8 mL/bag) in minimum essential medium–α, 10% FBS, and cytokines: thrombopoietin, interleukin (IL)-6, fms-like tyrosine kinase–3 ligand, and stem cell factor, each at a final concentration of 50 ng/mL, with or without NAM and incubated at 37°C in a humidified atmosphere of 5% CO2 in air.